At present, the scientific community has defined “stem cell” and its properties as follows. The stem cell refers to a cell with self-renewal and proliferation capability as well as the property of maintaining an undifferentiated status for a long time, and after the cell is induced and simulated appropriately, the cell can be differentiated into cell groups of different lineages and achieves a multi-differentiation of tissues with specific functions.
Based on the source and differentiation potential of the stem cell, the stem cell can be categorized into the following types:
1. Totipotent stem cell: It has the ability to be developed into a complete and independent living organism such as a zygote or an embryo and developed to a cell group of approximately eight cell stages.
2. Pluripotent stem cell: Approximately four days after a zygote is fertilized, the pluripotent stem cell starts entering into a blastocyst period, and the blastocyst can be divided into two parts: an outer layer of cells and an inner cell mass. In the development process of the embryo, the outer layer of cells will form placenta and fetus attached onto the required supporting tissues in the uterus, and the inner cell mass will form ectoderm, mesoderm and endoderm, each being divided into different systems and organs. Although the inner cell mass has the ability of forming various parts of human body, yet external supporting systems and environments for the growth of the placenta and fetus cannot be formed if the inner cell mass without the outer layer of cells is placed into the uterus of a healthy mature female, and a complete individual cannot be developed. Therefore, the multifunction ability still has partial limitations.
3. Multipotent stem cell: This is the most extensively studied stem cell, and the multipotent stem cell is differentiated downwardly from the pluripotent stem cell and capable of becoming the stem cell of specific tissues such as a hematopoietic stem cell and a mesenchymal stem cell, wherein the hematopoietic stem cell comes from peripheral blood, umbilical cord blood and marrow and can be divided into various blood corpuscles and lymphs, and the mesenchymal stem cell comes from adipose, periosteum, synovial membrane, marrow and mesenchymal tissue of a certain organ such as the placenta. For example, the hematopoietic stem cell can be a hematopoietic stem cell which can be differentiated into a lymph stem cell and a marrow stem cell, wherein the lymph stem cell can be differentiated into a lymphocyte, a killer cell, etc, and the bone marrow stem cell can be differentiated into a red blood corpuscle, a white blood corpuscle, a blood platelet, etc. The multipotent stem cell can be found in a body of both adult and child, and the self-regeneration ability of the stem cell plays an important role of supplying and refreshing the cells normally consumed in our body. At present, the already separated multipotent stem cells include brain, retina, bone marrow, liver, skeletal muscle, skin, umbilical cord, umbilical cord blood, adipose tissue, etc.
4. Unipotent stem cell: It generally refers to a stem cell with the ability of differentiating into a specific kind of tissue or is called a progenitor cell, and the cells of this sort generally exist in the tissues of each part of the body, and the most easily found stem cell is a liver progenitor cell, and nerve progenitor cell.
The mesenchymal stem cell was first academically defined as a colony-forming unit of fibroblast (CFU-Fs). In the culture process, a single layer of the mesenchymal stem cell is attached onto a surface of the plastic Petri dish, and in a form similar to fibroblast and in a spindle shape. This kind of stem cells will be proliferated rapidly in vitro to form a colony, and has a potential of differentiating into osteoblast, adipocyte, and chondrocyte. In recent years, researches indicate that this cell can be differentiated into hepatocyte, cardiomyocyte, neural cell, islet cell, etc (Minguell et al., 2001).
The source of the mesenchymal stem cell comes from the separation of various different tissues of a human body. For example, an adipose tissue obtained from direct surgical excision or obtained from liposuction is a rich source of stem cells, and the adipose tissue-derived stem cell (ADSCs) has the following advantages: low invasiveness, little harm to human body, high producing quantity at a time, and proliferation and culture in vitro, etc. In addition, the adipose tissue-derived stem cell also has the potential to differentiate into bone, cartridge, muscle and adipocyte (Zuk et al., 2002), so that the adipose tissue-derived stem cell is considered to be one of the stem cells with high development potential.
Regardless of fundamental medical research or clinical treatment, the research and application of stem cells require a sufficient number of cells and a culture in an appropriate environment including a stimulation such as an appropriate microenvironment or growth factor to prevent the stem cell from being aged before the proliferation and culture process, losing activity or differentiating into other cells. However, the difference including the cell separation technology, growth medium and culture condition causes a significant difference of proliferation and differentiation power of the stem cell (Pittenger et al., 2008). In addition, many journals reported the tendency of having an ageing issue of the mesenchymal stem cell in the culture and proliferation processes (Bonab et al., 2006; Shibata et al., 2007; Wagner et al., 2008). Therefore, the differences of performance and ageing of the cells in a proliferation of the stem cell probably hinder the clinical use of the mesenchymal stem cell. It is a major subject for scientist to culture the stem cell rapidly and effectively and amplify its number, while maintaining the undifferentiated status and reduce the ageing phenomenon, and having the multifunctional feature.
In recent years, researches reported and pointed out that a large quantity of paracrine factors such as a vascular endothelial growth factor (VEGF), an insulin-like growth factor 1 (IGF-I), an epidermal growth factor (EGF), a keratinocyte growth factor (KGF), angiopoietin-1, stromal-derived factor-1, macrophage inflammatory protein-1α, macrophage inflammatory protein-1β, and erythropoietin capable of promoting wound healing existed in a conditional medium of the cultured bone marrow mesenchymal stem cell (BMMSC) (Martin et al., 1997). The adipose tissue-derived stem cell (ADSC) has been shown that its gene expression and phenotype have no significant difference from those of the bone marrow mesenchymal stem cell, umbilical cord blood mesenchymal stem cell, periosteum mesenchymal stem cell, synoval mesenchymal stem cell and muscle mesenchymal stem cell (Sheehy et al., 2012; Hung et al., 2012; Hung et al., 2007). In view of the adipose tissue-derived stem cell has a better separation and in vitro proliferation condition than the bone marrow mesenchymal stem cell, and has a good chance to be applied in wound repair and regeneration. At present, the adipose tissue-derived stem cell in the conditional medium secretes the following growth factors, such as basic fibroblast growth factor (bFGF), keratinocyte growth factor (KGF), transformation growth factor (TGF-β), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), etc, and these growth factors may be related to wound healing. Therefore, it is a worthy research subject to proliferate mesenchymal stem cells and produce a large quantity of growth factors rapidly.
R.O.C. Pat. No. 201331366 entitled “In vitro serum-free somantic stem cell stem cell culture amplification technology” provides an in vitro serum-free somantic stem cell stem cell, and a method of plasma rich growth factor (PRGF) in serum-free stem cell culture liquid to perform primary culture and subculture in human somantic stem cell, and after the human somantic stem cell cultured by this method is subcultured for several times, the human somantic stem cell still maintains at a substantially undifferentiated status. However, the quantity of stem cells obtained by such culture method is approximately equal to 55,000 of cells/cm2 before the cells are subcultured to the third passage (P3). If it is necessary to obtain more stem cells, more culture days and more passages of subculture are required, thus the stem cells are exposed to more risk of contamination for a greater number of subcultures.
R. O. C. Pat. No. 201118172 entitled “Method of amplifying mesenchymal stem cells in low-density and low-oxygen culture” can increase human mesenchymal stem cells in the condition without affecting the cell proliferation to rapidly and effectively increase the proliferation in vitro, reduce the potential of ageing, and increase the potential of differentiation. However, the incubator of culturing cell used by most research organizations just provides the function of differentiating the pressure of carbon dioxide and adjusting the moisture. If it is necessary to culture in a low oxygen environment, the incubator with the oxygen pressure adjustment functions is relatively unaffordable by research organizations without sufficient research budgets.
R.O.C. Pat. No. 201231087, entitled “Manufacturing procedure of a maintenance product including a plurality of growth factors”, and the method comprises the steps of taking out a healthy adipose; mixing a predetermined volume of solvent into the adipose tissue to clean the adipose tissue; adding a predetermined dosage of reagent into a predetermined volume of adipose tissue and performing a centrifugal separation; the produced and separated adipose tissue is mixed into a predetermined dosage of enzyme into an eppendorf to vibrate the eppendorf; producing a precipitate for a cell culture to produce a somatic cell; and after obtaining the somatic cell, and the cell culture liquid produced by the aforementioned culture method contains a growth factor (such as VEGF, HGF, b-FGF, TGF, and IGF) for cell secretion to complete the raw material of high-performance of maintenance product. However, the culture method is to put the somatic cells into a basic medium DMEM (Dulbecco's Modified Eagle Medium) containing a 10% fetal bovine serum (10% FBS) for the culture, and the slow proliferation rate of somatic cells, so that the quantity of the secreted growth factor is relatively little. If it is necessary to obtain a large quantity of growth factor, a greater number of culture days and a greater number of passages of subculture must required, and thus the stem cells are exposed to more risk of contamination for a greater number of subcultures.
In summation of the description above, the multifunctional characteristic of the stem cell can be maintained to proliferate the in vitro human mesenchymal stem cells rapidly, while obtaining a large quantity of growth factor secreted from the human mesenchymal stem cells by using a human mesenchymal stem cells culture adjuvant and a culture method thereof, so as to rapidly and efficiently amplify the number of cells and obtain a large quantity of growth factors.